International Journal of Molecular Sciences Article Comparative Molecular Dynamics Investigation of the Electromotile Hearing Protein Prestin Gianfranco Abrusci 1,2,†, Thomas Tarenzi 1,3,†, Mattia Sturlese 4 , Gabriele Giachin 5 and Roberto Battistutta 5 and Gianluca Lattanzi 1,3,∗ 1 Department of Physics, University of Trento, Via Sommarive 14, 38123 Trento, Italy; [email protected] (G.A.); [email protected] (T.T.) 2 Cineca, Via Magnanelli 6/3, Casalecchio di Reno, 40033 Bologna, Italy 3 TIFPA-INFN, Via Sommarive 14, 38123 Trento, Italy 4 Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo 5, 35131 Padova, Italy; [email protected] 5 Department of Chemical Sciences, University of Padova, Via Marzolo 1, 35131 Padova, Italy; [email protected] (G.G.); [email protected] (R.B.) * Correspondence: [email protected] † These authors contributed equally to this work. Abstract: The mammalian protein prestin is expressed in the lateral membrane wall of the cochlear hair outer cells and is responsible for the electromotile response of the basolateral membrane, following hyperpolarisation or depolarisation of the cells. Its impairment marks the onset of severe diseases, like non-syndromic deafness. Several studies have pointed out possible key roles of residues located in the Transmembrane Domain (TMD) that differentiate mammalian prestins as incomplete transporters from the other proteins belonging to the same solute-carrier (SLC) superfamily, which Citation: Abrusci, G.; Tarenzi, T.; are classified as complete transporters. Here, we exploit the homology of a prototypical incomplete Sturlese, M.; Giachin, G.; Battistutta, transporter (rat prestin, rPres) and a complete transporter (zebrafish prestin, zPres) with target R.; Lattanzi, G. Comparative structures in the outward open and inward open conformations. The resulting models are then Molecular Dynamics Investigation of embedded in a model membrane and investigated via a rigorous molecular dynamics simulation the Electromotile Hearing Protein protocol. The resulting trajectories are analyzed to obtain quantitative descriptors of the equilibration Prestin. Int. J. Mol. Sci. 2021, 22, 8318. phase and to assess a structural comparison between proteins in different states, and between different https://doi.org/10.3390/ proteins in the same state. Our study clearly identifies a network of key residues at the interface ijms22158318 between the gate and the core domains of prestin that might be responsible for the conformational Academic Editor: Pere Garriga change observed in complete transporters and hindered in incomplete transporters. In addition, we study the pathway of Cl− ions in the presence of an applied electric field towards their putative Received: 29 June 2021 binding site in the gate domain. Based on our simulations, we propose a tilt and shift mechanism of Accepted: 26 July 2021 the helices surrounding the ion binding cavity as the working principle of the reported conformational Published: 2 August 2021 changes in complete transporters. Publisher’s Note: MDPI stays neutral Keywords: molecular dynamics simulations; SLC transporters; prestin; Non Linear Capacitance (NLC) with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Proteins in the solute-carrier (SLC) superfamily are active secondary transporters, whose study has been an active field of research in the last two decades [1–3]. Members Copyright: © 2021 by the authors. of the SLC superfamily, which is the second largest family of membrane proteins, play Licensee MDPI, Basel, Switzerland. crucial roles in a large number of physiological processes [4]; these range from the transport This article is an open access article of amino acids through the cell membrane (SLC1 family [5]), to the regulation of the distributed under the terms and extracellular concentration of neurotransmitters during synaptic activity (SLC6 family [6]), conditions of the Creative Commons to the pH regulation of blood [7]. The latter is the case of the SLC4A1 protein, also known Attribution (CC BY) license (https:// as band 3, which transports bicarbonate ions through the plasma membrane of erythrocytes creativecommons.org/licenses/by/ in an electroneutral exchange with chloride ions, a process fundamental for respiration [7]. 4.0/). Int. J. Mol. Sci. 2021, 22, 8318. https://doi.org/10.3390/ijms22158318 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 8318 2 of 19 Among the SLC protein families, of particular interest is the SLC26 one, whose members − − act as transporters of a broad range of substrates, including Cl , HCO3 , sulfate, oxalate, I−, and formate [8–12]. Mammalian prestin is an atypical member of the SLC26 family, since it shows no evidence of the transport of ligands across the membrane; instead, it works as a motor protein, and as such it has been extensively studied in the last decade [13–19]. Prestin consists of 744 amino acids, which can be divided in three major domains: the N-terminus (75 residues), the transmembrane sulfate transporter (TMD) domain (430 residues) and the anti-sigma factor antagonist (STAS) domain at the C-terminus (310 residues); both N- and C-termini are located in the cytoplasm. As a result of binding of an intracellular anion (chloride) to the TMD, the protein undergoes a conformational change that is sufficient to produce a significant increase of its size in the plane of the membrane. This electrome- chanical feedback mechanism, called electromotility, was discovered by Brownwell et al. in 1985 [20]. In humans, prestin is expressed in the lateral membrane wall of specialized auditory sensory cells, namely the cochlear outer hair cells (OHCs) [21,22]; the latter are found in the organ of Corti, the receptor organ of hearing, located in the inner ear. Here, prestin is densely packed in arrays, which elongate and contract in response to the hyperpolarisation and depolarisation of the surface of the membrane [23]. These elongations and contractions have a significant impact on the area of the basolateral membrane, with associated changes in surface area up to 4%; they occur in human cells at frequencies greater than 20 kHz [24] and in other mammalian species even faster, as in the case of the guinea pig’s OHCs, with changes occurring 120 microseconds after stimulation [25]. Such conformational transitions, originating electromotility, are responsible for the amplification of sound; mutations in human prestin impairing electromotility seem to lead to severe diseases, like non-syndromic deafness [26]. Electromotility of mammalian prestin is associated to a nonlinear dislocation of charges across the cell membrane, following stimulation from an external voltage—a phenomenon known as Non Linear Capacitance (NLC) [27]. This movement of charges can be explained by the relative motion of a charged moiety of the protein in the OHC lateral membrane or by a partial translocation of the ligand, namely the chloride ion. On the opposite, the membrane responds as a classical linear capacitor in the presence of non-mammalian ortholgs of prestin, which adhere to the function of the SLC26 family as antiporters, with bicarbonate ions being exchanged for chloride ions. These different mechanisms of actions are represented in Figure1. In response to an applied voltage, the membrane would exhibit NLC when containing a mammalian protein such as rat prestin, or an electric current when containing non-mammalian orthologs [14]. Mammalian prestin Non-mammalian prestin − Oxalate Cl Cl− Incomplete ion translocation + Complete ion translocation Elongation/contraction Figure 1. Schematic representations of the electromotile behaviour of prestin in mammals (blue), and of the full substrate transport process in non-mammalian prestin (orange). Int. J. Mol. Sci. 2021, 22, 8318 3 of 19 Numerous experimental studies have been dedicated to prestin and its orthologs. Some of them focused on the role of anions, in particular chloride, to trigger NLC [13,14]; other studies investigated how various mutations affect the electromotily and the transport of charges in the family members [17]. However, how prestin is able to give rise to the OHC elettromotility process is still unclear. The formation of oligomers (dimers or dimers of dimers) in the basolateral membrane seems important and, in this process, the STAS domain plays a fundamental role, as demonstrated by the structures of dimeric SLC26A9 [28]. However, the TMD domain of the single protomer is the basic unit for the translocation of anions [18]; since this (incomplete) ion transport is at the origin of NLC in prestin, we focused here on the TMD domain of the single protomer. Specifically, in order to investigate the different behaviour of prestin when expressed in mammals, or in one of its non-mammalian orthologs, we present here a computational comparative study of TMD from rat and zebrafish prestins (rPres and zPres, respectively), in two conformations: an inward open, with the putative binding site accessible to the solvent from the cytoplasmic side of the cell, and an outward open conformation, where the binding region is accessible from the exterior of the cell. Although the latter arrangement is not expected to be biologically relevant for the mammalian ortholog, its structural investigation is nonetheless informative on the differences between the two protein systems. Through our bioinformatics analyses and molecular dynamics (MD)